T cell receptors and immune therapy using the same

ABSTRACT

The present invention pertains to antigen recognizing constructs against COL6A3 antigens. The invention in particular provides novel T cell receptor (TCR) based molecules which are selective and specific for the tumor expressed antigen COL6A3. The TCR of the invention, and COL6A3 antigen binding fragments derived therefrom, are of use for the diagnosis, treatment and prevention of COL6A3 expressing cancerous diseases. Further provided are nucleic acids encoding the antigen recognizing constructs of the invention, vectors comprising these nucleic acids, recombinant cells expressing the antigen recognizing constructs and pharmaceutical compositions comprising the compounds of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.Nos. 62/376,059, filed 17 Aug. 2016 and 62/376,632, filed 18 Aug. 2016,and German Application No. 102016115246.3, filed 17 Aug. 2016, thecontent of each of these applications is herein incorporated byreference in their entirety.

This application also is related to PCT/EP2017/066630 filed 4 Jul. 2017,the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “2912919-074002_Sequence_Listing_ST25.txt” createdon 14 Aug. 2017, and 48,288 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention pertains to antigen recognizing constructs againstCOL6A3 antigens. The invention in particular provides novel T cellreceptor (TCR) based molecules which are selective and specific for thetumor expressed antigen COL6A3. The TCR of the invention, and COL6A3antigen binding fragments derived therefrom, are of use for thediagnosis, treatment and prevention of COL6A3 expressing cancerousdiseases. Further provided are nucleic acids encoding the antigenrecognizing constructs of the invention, vectors comprising thesenucleic acids, recombinant cells expressing the antigen recognizingconstructs and pharmaceutical compositions comprising the compounds ofthe invention.

BACKGROUND OF THE INVENTION

The collagens are a superfamily of proteins that play a role inmaintaining the integrity of various tissues. Collagens areextracellular matrix proteins and have a triple-helical domain as theircommon structural element. Collagen VI is a major structural componentof microfibrils. The basic structural unit of collagen VI is aheterotrimer of the alpha 1(VI), alpha 2(VI), and alpha 3(VI) collagenchains. The alpha 1(VI) and alpha 2(VI) chains are encoded by the COL6A1and COL6A2 genes, respectively. The protein encoded by the COL6A3 geneis the alpha 3 subunit of type VI collagen (alpha 3(VI) collagen chain)(Bertini et al., 2002 Eur. J. Paediatr. Neurol 6:193-8). COL6A3′ s geneexpression was previously shown to be associated with the progression ofbreast cancer and was elevated in colon cancer (Smith M J, et al.“Analysis of differential gene expression in colorectal cancer andstroma using fluorescence-activated cell sorting purification” Britishjournal of cancer. 2009; 100:1452-1464; Tilman G et al “Human periostingene expression in normal tissues, tumors and melanoma: evidences forperiostin production by both stromal and melanoma cells” Mol Cancer.2007; 6:80) and as a prognosis marker of colorectal carcinoma (Qiao J etal. “Stroma derived COL6A3 is a potential prognosis marker of colorectalcarcinoma revealed by quantitative proteomics” Oncotarget. 2015 Oct. 6;6(30): 29929-29946). COL6A3 gene locates 2q37 in the human genome andcontains 44 exons. The COL6A3 protein has 3177 amino acids and contains12 Von Willebrand factor type A (vWA) domains, one fibronectin type 3domain and one BPTI/Kunitz family of serine protease inhibitors (KU)domain.

T-cell based immunotherapy targets represent peptide epitopes derivedfrom tumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). These tumorassociated antigens (TAAs) can be peptides derived from all proteinclasses, such as enzymes, receptors, transcription factors, etc. whichare expressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defective ribosomal products (DRiPs) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

A TCR is a heterodimeric cell surface protein of the immunoglobulinsuper-family, which is associated with invariant proteins of the CD3complex involved in mediating signal transduction. TCRs exist in αβ andγδ forms, which are structurally similar but have quite distinctanatomical locations and probably functions. The extracellular portionof native heterodimeric αβTCR consists of two polypeptides, each ofwhich has a membrane-proximal constant domain, and a membrane-distalvariable domain. Each of the constant and variable domains includes anintra-chain disulfide bond. The variable domains contain the highlypolymorphic loops analogous to the complementarity determining regions(CDRs) of antibodies. The use of TCR gene therapy overcomes a number ofcurrent hurdles. It allows equipping patients' own T cells with desiredspecificities and generation of sufficient numbers of T cells in a shortperiod of time, avoiding their exhaustion. The TCR will be transducedinto central memory T cells or T cells with stem cell characteristics,which may ensure better persistence and function upon transfer.TCR-engineered T cells will be infused into cancer patients renderedlymphopenic by chemotherapy or irradiation, allowing efficientengraftment but inhibiting immune suppression.

SUMMARY OF THE INVENTION

While advances have been made in the development of molecular-targetingdrugs for cancer therapy, there remains a need in the art to develop newanti-cancer agents that specifically target molecules highly specific tocancer cells. The present description addresses that need by providingnovel COL6A3 TCRs, respective recombinant TCR constructs, nucleic acids,vectors and host cells that specifically bind COL6A3 epitope(s) asdisclosed; and methods of using such molecules in the treatment ofcancer.

The object of the invention is solved in a first aspect by an antigenrecognizing construct comprising at least one complementary determiningregion (CDR) 3 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,or preferably 100% sequence identity to an amino acid sequence selectedfrom SEQ ID Nos. 3, 9, 15, 21, 27 and 33.

In another additional or alternative embodiment, the antigen recognizingconstruct may further comprise a CDR1 and/or a CDR2 domain sequence.Within the variable domain, CDR1 and CDR2 are found in the variable (V)region of a polypeptide chain, and CDR3 includes some of V, all ofdiversity (D) and joining (J) regions. CDR3 is the most variable and isthe main CDR responsible for specifically and selectively recognizing anantigen. CDR1 and CDR2 sequences may be selected from a CDR sequence ofa human variable chain allele.

Native alpha-beta heterodimeric TCRs have an alpha chain and a betachain. Each chain comprises variable, joining and constant regions, andthe beta chain also usually contains a short diversity region betweenthe variable and joining regions, but this diversity region is oftenconsidered as part of the joining region. Each variable region comprisesthree CDRs (Complementarity Determining Regions) embedded in a frameworksequence, one being the hypervariable region named CDR3. There areseveral types of alpha chain variable (Vα) regions and several types ofbeta chain variable (Vβ) regions distinguished by their framework, CDR1and CDR2 sequences, and by a partly defined CDR3 sequence. The Vα typesare referred to in IMGT nomenclature by a unique TRAV number, Vβ typesare referred to by a unique TRBV number.

Therefore, in one additional or alternative embodiment the antigenrecognizing construct of the invention comprises CDR1, CDR2 and CDR3sequences in a combination as provided in table 1 herein below, whichdisplay the respective variable chain allele together with the CDR3sequence. Therefore, preferred are antigen recognizing constructs of theinvention which comprise at least one, preferably, all three CDRsequences CDR1, CDR2 and CDR3. Preferably, an antigen recognizingconstruct of the invention comprises the respective CDR1 to CDR3 of oneindividual herein disclosed TCR variable region of the invention.

The term “specificity” or “antigen specificity” or “specific for” agiven antigen, as used herein means that the antigen recognizingconstruct can specifically bind to said antigen, preferably a COL6A3antigen, more preferably with high avidity, when said antigen is by HLA,preferably HLA A2. For example, a TCR, as antigen recognizing construct,may be considered to have “antigenic specificity” for COL6A3 antigens,if T cells expressing the TCR and contacted with a COL6A3 presenting HLAsecrete at least about 200 pg/ml or more (e.g., 250 pg/ml or more, 300pg/ml or more, 400 pg/ml or more, 500 pg/ml or more, 600 pg/ml or more,700 pg/ml or more, 1000 pg ml or more, 2,000 pg/ml or more, 2,500 pg/mlor more, 5,000 pg/ml or more) of interferon γ (IFN-γ) upon co-culturewith target cells pulsed with a low concentration of a COL6A3 antigen,such as the COL6A3 epitopes and antigens provided herein below (e.g.,about 10-11 mol/1, 10-10 mol/1, 10-9 mol/1, 10-8 mol/1, 10-7 mol/1, 10-6mol/1, 10-5 mol/1). Alternatively, or additionally, a TCR may beconsidered to have “antigenic specificity” for COL6A3, if T cellsexpressing the TCR secrete at least twice as much IFN-γ as theuntransduced background level of IFN-γ upon co-culture with target cellspulsed with a low concentration of COL6A3 antigens. Such a “specificity”as described above can—for example—be analyzed with an ELISA.

In one alternative or additional embodiment of the invention, theantigen recognizing construct selectively binds to a COL6A3 antigen;preferably wherein the COL6A3 antigen is a protein epitope or peptidehaving an amino acid sequence shown in SEQ ID NO: 58 to 67, mostpreferably SEQ ID NO: 58, or a variant thereof, wherein the variant isan amino acid deletion, addition, insertion or substitution of not morethan three, preferably two and most preferably not more than one aminoacid position.

The term “selectivity” or “selective recognizing/binding” is understoodto refer to the property of an antigen recognizing construct, such as aTCR or antibody, to selectively recognize or bind to preferably only onespecific epitope and preferably shows no or substantially nocross-reactivity to another epitope. Preferably “selectivity” or“selective recognizing/binding” means that the antigen recognizingconstruct (e.g. a TCR) selectively recognizes or binds to preferablyonly one specific epitope and preferably shows no or substantially nocross-reactivity to another epitope, wherein said epitope is unique forone protein, such that the antigen recognizing construct shows no orsubstantially no cross-reactivity to another epitope and anotherprotein.

The antigen recognizing construct according to the invention ispreferably selected from an antibody, or derivative or fragment thereof,or a T cell receptor (TCR), or derivative or fragment thereof. Aderivative or fragment of an antibody or TCR of the invention shallpreferably retain the antigen binding/recognizing ability of the parentmolecule, in particular its specificity and/or selectivity as explainedabove. Such binding functionality may be retained by the presence of aCDR3 region as defined herein.

In an embodiment of the invention, the inventive TCRs are able torecognize COL6A3 antigens in a major histocompatibility complex (MHC)class I-dependent manner. “MHC class I-dependent manner,” as usedherein, means that the TCR elicits an immune response upon binding toCOL6A3 antigens within the context of an MHC class I molecule. The MHCclass I molecule can be any MHC class I molecule known in the art, e.g.,HLA-A molecules. In a preferred embodiment of the invention, the MHCclass I molecule is an HLA-A2 molecule.

The invention provides both single chain antigen recognizing constructand double chain recognizing constructs.

The invention in particular provides a TCR as antigen recognizingconstruct, or fragment or derivative thereof. The TCR preferably is ofhuman, which is understood as being generated from a human TCR locus andtherefore comprising human TCR sequences. Furthermore, the TCR of theinvention may be characterized in that it is of human origin andspecifically recognizes a COL6A3 antigen.

Another embodiment of the invention additionally or alternativelyprovides the antigen recognizing construct described above, whichinduces an immune response, preferably wherein the immune response ischaracterized by an increase in interferon (IFN) γ levels.

TCRs of the invention may be provided as single chain α or β or γ and δ,molecules, or alternatively as double chain constructs composed of boththe α and β chain, or γ and δ chain. The antigen recognizing constructof the invention may comprise a TCR α or γ chain; and/or a TCR β or δchain; wherein the TCR α or γ chain comprises a CDR3 having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to anamino acid sequence selected from SEQ ID Nos. 3, 15, and 27, and/orwherein the TCR β or δ chain comprises a CDR3 having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID Nos. 9, 21, and 33.

Most preferably, in some additional embodiments, wherein the disclosurerefers to antigen recognizing constructs comprising any one, two or allof the CDR1 to CDR3 regions of the herein disclosed TCR chains (seetable 1), such antigen recognizing constructs may be preferred, whichcomprise the respective CDR sequence having three, two, and preferablyonly one, modified amino acid residues. A modified amino acid residuemay be selected from an amino acid insertion, deletion or substitution.Most preferred is that the three, two, preferably one modified aminoacid residue is the first or last amino acid residue of the respectiveCDR sequence. If the modification is a substitution then it ispreferable in some embodiments that the substitution is a conservativeamino acid substitution.

If the antigen recognizing construct of the invention is composed of atleast two amino acid chains, such as a double chain TCR, or antigenbinding fragment thereof, the antigen recognizing construct maycomprises in a first polypeptide chain the amino acid sequence accordingto SEQ ID NO: 3, and in a second polypeptide chain the amino acidsequence according to SEQ ID NO: 9; or in a first polypeptide chain theamino acid sequence according to SEQ ID NO: 15, and in a secondpolypeptide chain the amino acid sequence according to SEQ ID NO: 21; orin a first polypeptide chain the amino acid sequence according to SEQ IDNO: 27, and in a second polypeptide chain the amino acid sequenceaccording to SEQ ID NO: 33. Any one of the aforementioned double chainTCR, or antigen binding fragments thereof, are preferred TCR of thepresent invention. In some embodiments, the CDR3 of the double chain TCRof the invention may be mutated. Mutations of the CDR3 sequences of SEQID NOs: 9 to 28 as provided above preferably include a substitution,deletion, addition, or insertion of not more than three, preferably two,and most preferably not more than one amino acid residue. In someembodiments, the first polypeptide chain may be a TCR α or γ chain, andthe second polypeptide chain may be a TCR β or δ chain. Preferred is thecombination of an αβ or γδ TCR.

The TCR, or the antigen binding fragment thereof, is in some embodimentscomposed of a TCR α and a TCR β chain, or γ and δ chain. Such a doublechain TCR comprises within each chain variable regions, and the variableregions each comprise one CDR1, one CDR2 and one CDR3 sequence. The TCRscomprises the CDR1 to CDR3 sequences as comprised in the variable chainamino acid sequence of SEQ ID NO: 4 and SEQ ID NO: 10 (R4P1D10), or SEQID NO: 16 and SEQ ID NO: 22 (R4P3F9); or SEQ ID NO: 28 and SEQ ID NO: 34(R4P3H3).

Some embodiments of the invention pertain to a TCR, or a fragmentthereof, composed of a TCR α and a TCR β chain, wherein said TCRcomprises the variable region sequences having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or preferably 100% sequence identity to theamino acid sequence selected from the α and β chain according to SEQ IDNO: 4 and 10 respectively, or 16 and 22 respectively; or 28 and 34respectively.

The inventive TCRs may further comprise a constant region derived fromany suitable species, such as any mammal, e.g., human, rat, monkey,rabbit, donkey, or mouse. In an embodiment of the invention, theinventive TCRs further comprise a human constant region. In somepreferred embodiments, the constant region of the TCR of the inventionmay be slightly modified, for example, by the introduction ofheterologous sequences, preferably mouse sequences, which may increaseTCR expression and stability.

Some embodiments of the invention pertain to a TCR, or a fragmentthereof, composed of a TCR α and a TCR β chain, wherein said TCRcomprises the constant region having at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or preferably 100% sequence identity to an amino acidsequence selected from of the α and β chain according to SEQ ID NO: 5and 11 respectively, or 17 and 23 respectively; or 29 and 35respectively.

The TCR α or γ chain of the invention may further comprise a CDR1 havingat least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequenceidentity to an amino acid sequence selected from SEQ ID Nos. 1, 13, and25; and/or a CDR2 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%,99%, or 100% sequence identity to an amino acid sequence selected fromSEQ ID Nos. 2, 14, and 26.

According to the invention the TCR β or δ chain may further comprise aCDR1 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%sequence identity to an amino acid sequence selected from SEQ ID Nos. 7,19, and 31; and/or a CDR2 having at least 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or 100% sequence identity to an amino acid sequence selectedfrom SEQ ID Nos. 8, 20, and 32.

The antigen recognizing construct may in a further embodiment comprise abinding fragment of a TCR, and wherein said binding fragment comprisesCDR1 to CDR3, optionally selected from the CDR1 to CDR3 sequences havingthe amino acid sequences of SEQ ID Nos. 1, 2, 3, or 7, 8, 9 or 13, 14,15, or 19, 20, 21, or 25, 26, 27 or 31, 32, 33.

In further embodiments of the invention the antigen recognizingconstruct as described herein before is a TCR, or a fragment thereof,composed of at least one TCR α and one TCR β chain sequence, whereinsaid TCR α chain sequence comprises the CDR1 to CDR3 sequences havingthe amino acid sequences of SEQ ID NO: 1 to 3, and said TCR β chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NO: 7 to 9; or wherein said TCR α chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NO: 13 to 15, and said TCR β chain sequence comprises the CDR1 toCDR3 sequences having the amino acid sequences of SEQ ID NO: 19 to 21;or wherein said TCR α chain sequence comprises the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NO: 25 to 27, andsaid TCR β chain sequence comprises the CDR1 to CDR3 sequences havingthe amino acid sequences of SEQ ID NO: 31 to 33.

In further embodiments of the invention the antigen recognizingconstruct as described herein before is a TCR, or a fragment thereof,comprising at least one TCR α and one TCR β chain sequence, wherein saidTCR α chain sequence comprises a variable region sequence having theamino acid sequence of SEQ ID No. 4, and wherein said TCR β chainsequence comprises a variable region sequence having the amino acidsequence of SEQ ID No. 10; or wherein said TCR α chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 16, and wherein said TCR β chain sequence comprises avariable region sequence having the amino acid sequence of SEQ ID No.22; or wherein said TCR α chain sequence comprises a variable regionsequence having the amino acid sequence of SEQ ID No. 28, and whereinsaid TCR β chain sequence comprises a variable region sequence havingthe amino acid sequence of SEQ ID No. 34.

In further embodiments of the invention the antigen recognizingconstruct as described herein before is a TCR, or a fragment thereof,further comprising a TCR constant region having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID Nos. 5, 11, 17, 23, 29 and 35, preferablywherein the TCR is composed of at least one TCR α and one TCR β chainsequence, wherein the TCR α chain sequence comprises a constant regionhaving at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequenceidentity to an amino acid sequence selected from SEQ ID Nos. 5, 17, and29.

Also disclosed are antigen recognizing constructs as described hereinbefore comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID No. 6, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID No. 12. The invention also provides TCRs comprising afirst TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID No. 18,and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNo. 24. In further embodiments the invention provides antigenrecognizing constructs which are TCR and comprise a first TCR chainhaving at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID No. 30, and a second TCRchain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID No. 36.

As used herein, the term “murine” or “human,” when referring to anantigen recognizing construct, or a TCR, or any component of a TCRdescribed herein (e.g., complementarity determining region (CDR),variable region, constant region, α chain, and/or β chain), means a TCR(or component thereof), which is derived from a mouse or a humanunrearranged TCR locus, respectively.

In an embodiment of the invention, chimeric TCR are provided, whereinthe TCR chains comprise sequences from multiple species. Preferably, aTCR of the invention may comprise an α chain comprising a human variableregion of an α chain and, for example, a murine constant region of amurine TCR α chain.

In one embodiment, the TCR of the invention is a human TCR comprisinghuman variable regions according to the above embodiments and humanconstant regions.

The TCR of the invention may be provided as a single chain TCR (scTCR).A scTCR can comprise a polypeptide of a variable region of a first TCRchain (e.g., an alpha chain) and a polypeptide of an entire(full-length) second TCR chain (e.g., a beta chain), or vice versa.Furthermore, the scTCR can optionally comprise one or more linkers whichjoin the two or more polypeptides together. The linker can be, forinstance, a peptide, which joins together two single chains, asdescribed herein. Also provided is such a scTCR of the invention, whichis fused to a human cytokine, such as IL-2, IL-7 or IL-15.

The antigen recognizing construct according to the invention can also beprovided in the form of a multimeric complex, comprising at least twoscTCR molecules, wherein said scTCR molecules are each fused to at leastone biotin moiety, and wherein said scTCRs are interconnected bybiotin-streptavidin interaction to allow the formation of saidmultimeric complex. Similar approaches for the generation of multimericTCR are also possible and included in this disclosure. Also provided aremultimeric complexes of a higher order, comprising more than two scTCRof the invention.

For the purposes of the present invention, a TCR is a moiety having atleast one TCR alpha or gamma and/or TCR beta or delta variable domain.Generally, they comprise both a TCR alpha variable domain and a TCR betavariable domain. They may be αβ heterodimers or may be in single chainformat. For use in adoptive therapy, an αβ heterodimeric TCR may, forexample, be transfected as full length chains having both cytoplasmicand transmembrane domains. If desired, an introduced disulfide bondbetween residues of the respective constant domains may be present.

In a preferred embodiment, the antigen recognizing construct is a humanTCR, or fragment or derivative thereof. A human TCR or fragment orderivative thereof is a TCR, which comprises over 50% of thecorresponding human TCR sequence. Preferably, only a small part of theTCR sequence is of artificial origin or derived from other species. Itis known, however, that chimeric TCRs, e.g. derived from human originwith murine sequences in the constant domains, are advantageous.Particularly preferred are, therefore, TCRs in accordance with thepresent invention, which contains murine sequences in the extracellularpart of their constant domains.

Thus, it is also preferred that the inventive antigen recognizingconstruct is able to recognize its antigen in a human leucocyte antigen(HLA) dependent manner, preferably in a HLA-A02 dependent manner. Theterm “HLA dependent manner” in the context of the present inventionmeans that the antigen recognizing construct binds to the antigen onlyin the event that the antigenic peptide is presented by said HLA.

The antigen recognizing construct in accordance with the invention inone embodiment preferably induces an immune response, preferably whereinthe immune response is characterized by the increase in interferon (IFN)γ levels.

Also provided by the invention is a polypeptide comprising a functionalportion of any of the TCRs (or functional variants thereof) describedherein, for examples, of any one of the TCRs selected from R4P1D10,R4P3F9, and R4P3H3, as provided in the example section and table 1. Theterm “polypeptide” as used herein includes oligopeptides and refers to asingle chain of amino acids connected by one or more peptide bonds. Withrespect to the inventive polypeptides, the functional portion can be anyportion comprising contiguous amino acids of the TCR (or functionalvariant thereof), of which it is a part, provided that the functionalportion specifically binds to a COL6A3 antigen, preferably as disclosedherein in table 2, and peptides A1 to A9 (SEQ ID NOs: 59-67). The term“functional portion” when used in reference to a TCR (or functionalvariant thereof) refers to any part or fragment of the TCR (orfunctional variant thereof) of the invention, which part or fragmentretains the biological activity of the TCR (or functional variantthereof), of which it is a part (the parent TCR or parent functionalvariant thereof). Functional portions encompass, for example, thoseparts of a TCR (or functional variant thereof) that retain the abilityto specifically bind to a COL6A3 antigen (in an HLA-A2-dependentmanner), or detect, treat, or prevent cancer, to a similar extent, thesame extent, or to a higher extent, as the parent TCR (or functionalvariant thereof). In reference to the parent TCR (or functional variantthereof), the functional portion can comprise, for instance, about 10%,25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent TCR variablesequences (or functional variant thereof).

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, in whichadditional amino acids are not found in the amino acid sequence of theparent TCR or functional variant thereof. Desirably, the additionalamino acids do not interfere with the biological function of thefunctional portion, e.g., specifically binding to COL6A3 antigens;and/or having the ability to detect cancer, treat or prevent cancer,etc. More desirably, the additional amino acids enhance the biologicalactivity, as compared to the biological activity of the parent TCR orfunctional variant thereof.

The polypeptide can comprise a functional portion of either or both ofthe α and β chains of the TCRs or functional variant thereof of theinvention, such as a functional portion comprising one of more of CDR1,CDR2, and (preferably) CDR3 of the variable region(s) of the α chainand/or β chain of a TCR or functional variant thereof of the invention.In an embodiment of the invention, the polypeptide can comprise afunctional portion comprising the amino acid sequence of SEQ ID NO: 3,9, 15, 21, 27, and 33 (CDR3 of the variable regions of the TCR of theinvention), or a combination thereof. In an embodiment of the invention,the inventive polypeptide can comprise, for instance, the variableregion of the inventive TCR or functional variant thereof comprising acombination of the CDR regions set forth above. In this regard, thepolypeptide can comprise the amino acid sequence of any of SEQ ID NO: 4,10, 16, 22, 28, and 34 (the variable regions of an α or β chain of theTCR of the invention).

In some instances, the construct of the invention may comprise one ortwo polypeptide chains comprising a sequences according to any of theSEQ ID NO: 1 to 36 (CDR sequences, constant and variable regions andfull length sequences), or functional fragments thereof, and furthercomprise(s) other amino acid sequences, e.g., an amino acid sequenceencoding an immunoglobulin or a portion thereof, then the inventiveprotein can be a fusion protein. In this regard, the invention alsoprovides a fusion protein comprising at least one of the inventivepolypeptides described herein along with at least one other polypeptide.The other polypeptide can exist as a separate polypeptide of the fusionprotein, or can exist as a polypeptide, which is expressed in frame (intandem) with one of the inventive polypeptides described herein. Theother polypeptide may include any peptidic or proteinaceous molecule, ora portion thereof, including, but not limited to an immunoglobulin, CD3,CD4, CD8, an MHC molecule, a CD1 molecule, e.g., CD1a, CD1b, CD1c, CD1d,etc.

The fusion protein can comprise one or more copies of the inventivepolypeptide and/or one or more copies of the other polypeptide. Forinstance, the fusion protein can comprise 1, 2, 3, 4, 5, or more, copiesof the inventive polypeptide and/or of the other polypeptide. Suitablemethods of making fusion proteins are known in the art, and include, forexample, recombinant methods. In some embodiments of the invention, theTCRs (and functional portions and functional variants thereof),polypeptides, and proteins of the invention may be expressed as a singleprotein comprising a linker peptide linking the α chain and the β chain,and linking the γ chain and the δ chain. In this regard, the TCRs (andfunctional variants and functional portions thereof), polypeptides, andproteins of the invention comprising the amino acid sequences of thevariable regions of the TCR of the invention and may further comprise alinker peptide. The linker peptide may advantageously facilitate theexpression of a recombinant TCR (including functional portions andfunctional variants thereof), polypeptide, and/or protein in a hostcell. The linker peptide may comprise any suitable amino acid sequence.Linker sequences for single chain TCR constructs are well known in theart. Such a single chain construct may further comprise one, or two,constant domain sequences. Upon expression of the construct includingthe linker peptide by a host cell, the linker peptide may also becleaved, resulting in separated α and β chains, and separated γ and δchain.

As already mentioned above, the binding functionality of the TCR of theinvention may be provided in the framework of an antibody. The term“antibody” in its various grammatical forms is used herein to refer toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain anantigen-binding site or a paratope. Such molecules are also referred toas “antigen binding fragments” of immunoglobulin molecules. Theinvention further provides an antibody, or antigen binding portionthereof, which specifically binds to the antigens described herein. Theantibody can be any type of immunoglobulin that is known in the art. Forinstance, the antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG,IgM, etc. The antibody can be monoclonal or polyclonal. The antibody canbe a naturally-occurring antibody, e.g., an antibody isolated and/orpurified from a mammal, e.g., mouse, rabbit, goat, horse, chicken,hamster, human, etc. Alternatively, the antibody can be agenetically-engineered antibody, e.g., a humanized antibody or achimeric antibody. The antibody can be in monomeric or polymeric form.

The invention also provides antigen binding portions of any of theantibodies described herein. The antigen binding portion can be anyportion that has at least one antigen binding site, such as Fab,F(ab′)2, dsFv, sFv, diabodies, and triabodies. A single-chain variableregion fragment (sFv) antibody fragment, which consists of a truncatedFab fragment comprising the variable (V) domain of an antibody heavychain linked to a V domain of a light antibody chain via a syntheticpeptide, can be generated using routine recombinant DNA technologytechniques. Similarly, disulfide-stabilized variable region fragments(dsFv) can be prepared by recombinant DNA technology, antibody fragmentsof the invention, however, are not limited to these exemplary types ofantibody fragments. Also, the antibody, or antigen binding portionthereof, can be modified to comprise a detectable label, such as, forinstance, a radioisotope, a fluorophore (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkalinephosphatase, horseradish peroxidase), and element particles (e.g., goldparticles). In some instances, the TCR CDR3 sequence may be slightlymodified, but preferably by not more than 3 amino acid residues,preferably only two and most preferably only one amino acid position, ascompared to the CDR3 sequences provided in SEQ ID Nos: 3, 9, 15, 21, 27,and 33. Preferably, the antibodies comprise the CDR3, preferably all ofCDR1 to CDR3 regions in the combination, as indicated for the TCR of theinvention in table 1.

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Kohler andMilstein, Eur. J. Immunol, 5, 51 1-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds.), Immunobiology, 8 Ed., Garland Publishing, New York, N.Y.(2011)). Alternatively, other methods, such as EBV-hybridoma methods(Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), andRoder et al, Methods Enzymol, 121, 140-67 (1986)), and bacteriophagevector expression systems (see, e.g., Huse et al., Science, 246, 1275-81(1989)) are known in the art. Further, methods of producing antibodiesin non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806,5,569,825, and 5,714,352, and U.S. Patent Application Publication No.2002/0197266.

Some embodiments of the invention also pertain to TCRs, or functionalfragments and polypeptides thereof, which are soluble TCRs. As usedherein, the term “soluble T-cell receptor” refers to heterodimerictruncated variants of native TCRs, which comprise extracellular portionsof the TCR α-chain and β-chain linked by a disulfide bond, but whichlack the transmembrane and cytosolic domains of the native protein. Theterms “soluble T-cell receptor α-chain sequence and soluble T-cellreceptor β-chain sequence” refer to TCR α-chain and β-chain sequencesthat lack the transmembrane and cytosolic domains. The sequence (aminoacid or nucleic acid) of the soluble TCR α-chain and β-chains may beidentical to the corresponding sequences in a native TCR or may comprisevariant soluble TCR α-chain and β-chain sequences, as compared to thecorresponding native TCR sequences. The term “soluble T-cell receptor”as used herein encompasses soluble TCRs with variant or non-variantsoluble TCR α-chain and β-chain sequences. The variations may be in thevariable or constant regions of the soluble TCR α-chain and β-chainsequences and can include, but are not limited to, amino acid deletion,insertion, substitution mutations as well as changes to the nucleic acidsequence, which do not alter the amino acid sequence. Soluble TCR of theinvention in any case retain the binding functionality of their parentmolecules.

The above problem is further solved by a nucleic acid encoding for anantigen recognizing construct of the invention, or any of theaforementioned protein or polypeptide constructs. The nucleic acidpreferably (a) has a strand encoding for an antigen recognizingconstruct according to the invention; (b) has a strand complementary tothe strand in (a); or (c) has a strand that hybridizes under stringentconditions with a molecule as described in (a) or (b). Stringentconditions are known to the person of skill in the art, specificallyfrom Sambrook et al, “Molecular Cloning”. In addition to that, thenucleic acid optionally has further sequences, which are necessary forexpressing the nucleic acid sequence corresponding to the protein,specifically for expression in a mammalian/human cell. The nucleic acidused can be contained in a vector suitable for allowing expression ofthe nucleic acid sequence corresponding to the peptide in a cell.However, the nucleic acids can also be used to transform anantigen-presenting cell, which may not be restricted to classicalantigen-presenting cells, such as dendritic cells, in such a way thatthey themselves produce the corresponding proteins on their cellularsurface.

By “nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication. The nucleic acid can compriseany nucleotide sequence, which encodes any of the TCRs, polypeptides, orproteins, or functional portions or functional variants thereofdescribed herein.

Furthermore, the invention provides a vector comprising a nucleic acidin accordance to the invention as described above. Desirably, the vectoris an expression vector or a recombinant expression vector. The term“recombinant expression vector” refers in context of the presentinvention to a nucleic acid construct that allows for the expression ofan mRNA, protein or polypeptide in a suitable host cell. The recombinantexpression vector of the invention can be any suitable recombinantexpression vector, and can be used to transform or transfect anysuitable host. Suitable vectors include those designed for propagationand expansion or for expression or both, such as plasmids and viruses.Examples of animal expression vectors include pEUK-Cl, pMAM, andpMAMneo. Preferably, the recombinant expression vector is a viralvector, e.g., a retroviral vector. The recombinant expression vectorcomprises regulatory sequences, such as transcription and translationinitiation and termination codons, which are specific to the type ofhost cell (e.g., bacterium, fungus, plant, or animal), into which thevector is to be introduced and in which the expression of the nucleicacid of the invention may be performed. Furthermore, the vector of theinvention may include one or more marker genes, which allow forselection of transformed or transfected hosts. The recombinantexpression vector can comprise a native or normative promoter operablylinked to the nucleotide sequence encoding the constructs of theinvention, or to the nucleotide sequence, which is complementary to orwhich hybridizes to the nucleotide sequence encoding the constructs ofthe invention. The selections of promoters include, e.g., strong, weak,inducible, tissue-specific and developmental-specific promoters. Thepromoter can be a non-viral promoter or a viral promoter. The inventiverecombinant expression vectors can be designed for either transientexpression, for stable expression, or for both. Also, the recombinantexpression vectors can be made for constitutive expression or forinducible expression.

The invention also pertains to a host cell comprising an antigenrecognizing construct in accordance with the invention. Specifically,the host cell of the invention comprises a nucleic acid, or a vector asdescribed herein above. The host cell can be a eukaryotic cell, e.g.,plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g.,bacteria or protozoa. The host cell can be a cultured cell or a primarycell, i.e., isolated directly from an organism, e.g., a human. The hostcell can be an adherent cell or a suspended cell, i.e., a cell thatgrows in suspension. For purposes of producing a recombinant TCR,polypeptide, or protein, the host cell is preferably a mammalian cell.Most preferably, the host cell is a human cell. While the host cell canbe of any cell type, can originate from any type of tissue, and can beof any developmental stage, the host cell preferably is a peripheralblood leukocyte (PBL) or a peripheral blood mononuclear cell (PBMC).More preferably, the host cell is a T cell. The T cell can be any Tcell, such as a cultured T cell, e.g., a primary T cell, or a T cellfrom a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cellobtained from a mammal, preferably a T cell or T cell precursor from ahuman patient. If obtained from a mammal, the T cell can be obtainedfrom numerous sources, including but not limited to blood, bone marrow,lymph node, the thymus, or other tissues or fluids. T cells can also beenriched for or purified. Preferably, the T cell is a human T cell. Morepreferably, the T cell is a T cell isolated from a human. The T cell canbe any type of T cell and can be of any developmental stage, includingbut not limited to, CD4-positive and/or CD8-positive, CD4-positivehelper T cells, e.g., Th1 and Th2 cells, CD8-positive T cells (e.g.,cytotoxic T cells), tumor infiltrating cells (TILs), memory T cells,naive T cells, and the like. Preferably, the T cell is a CD8-positive Tcell or a CD4-positive T cell.

Preferably, the host cell of the invention is a lymphocyte, preferably,a T lymphocyte, such as a CD4-positive or CD8-positive T-cell. The hostcell furthermore preferably is a tumor reactive T cell specific forCOL6A3 expressing tumor cells.

One further aspect of the present invention relates to the hereindisclosed antigen recognizing constructs, nucleic acids, vectors,pharmaceutical compositions and/or host cell for use in medicine. Theuse in medicine in one preferred embodiment includes the use in thediagnosis, prevention and/or treatment of a tumor disease, such as amalignant or benign tumor disease. The tumor disease is, for example, atumor disease characterized by the expression of COL6A3, in a cancer ortumor cell of said tumor disease.

With respect to the above mentioned medical applications of the antigenrecognizing constructs and other materials derived therefrom, the to betreated and/or diagnosed cancer can be any cancer, including any ofacute lymphocytic cancer, acute myeloid leukemia, alveolarrhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer ofthe anus, anal canal, or anorectum, cancer of the eye, cancer of theintrahepatic bile duct, cancer of the joints, cancer of the neck,gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear,cancer of the oral cavity, cancer of the vagina, cancer of the vulva,chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer,esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor,glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynxcancer, liver cancer, lung cancer, malignant mesothelioma, melanoma,multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer ofthe oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer,peritoneum, omentum, and mesentery cancer, pharynx cancer, prostatecancer, rectal cancer, renal cancer, skin cancer, small intestinecancer, soft tissue cancer, stomach cancer, testicular cancer, thyroidcancer, cancer of the uterus, ureter cancer, and urinary bladder cancer.A preferred cancer is cancer is cancer of the uterine cervix,oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis. Aparticularly preferred cancer is a COL6A3 positive cancer, includinggastrointestinal and gastric cancer.

The constructs, proteins, TCRs antibodies, polypeptides and nucleicacids of the invention are in particular for use in immune therapy,preferably, in adoptive T cell therapy. The administration of thecompounds of the invention can, for example, involve the infusion of Tcells of the invention into said patient. Preferably, such T cells areautologous T cells of the patient and in vitro transduced with a nucleicacid or antigen recognizing construct of the present invention.

WO 2016/011210 discloses engineered cells for adoptive therapy,including NK cells and T cells, and compositions containing the cells,and methods for their administration to subjects. The cells can containgenetically engineered antigen receptors that specifically bind toantigens, such as chimeric antigen receptors (CARs) and costimulatoryreceptors.

The objective of the invention is also solved by a method ofmanufacturing a COL6A3 specific antigen recognizing construct expressingcell line, comprising

-   a. Providing a suitable host cell,-   b. Providing a genetic construct comprising a coding sequence    encoding for an antigen recognizing construct according to the    herein disclosed invention,-   c. Introducing into said suitable host cell said genetic construct,    and-   d. Expressing said genetic construct by said suitable host cell.

The method may further comprise a step of cell surface presentation ofsaid antigen recognizing construct on said suitable host cell.

In other preferred embodiments, the genetic construct is an expressionconstruct comprising a promoter sequence operably linked to said codingsequence.

Preferably, said antigen recognizing construct is of mammalian origin,preferably of human origin. The preferred suitable host cell for use inthe method of the invention is a mammalian cell, such as a human cell,in particular a human T lymphocyte. T cells for use in the invention aredescribed in detail herein above.

Also encompassed by the invention are embodiments, wherein said antigenrecognizing construct is a modified TCR, wherein said modification isthe addition of functional domains, such as a label or a therapeuticallyactive substance. Furthermore, encompassed are TCR having alternativedomains, such as an alternative membrane anchor domain instead of theendogenous transmembrane region.

Desirably, the transfection system for introducing the genetic constructinto said suitable host cell is a retroviral vector system. Such systemsare well known to the skilled artisan.

Also comprised by the present invention is in one embodiment theadditional method step of isolation and purification of the antigenrecognizing construct from the cell and, optionally, the reconstitutionof the translated antigen recognizing construct-fragments in a T-cell.

In an alternative aspect of the invention a T-cell is provided obtainedor obtainable by a method for the production of a T cell receptor (TCR),which is specific for tumorous cells and has high avidity as describedherein above. Such a T cell is depending on the host cell used in themethod of the invention, for example, a human or non-human T-cell,preferably a human TCR.

The inventive TCRs, polypeptides, proteins, (including functionalvariants thereof), nucleic acids, recombinant expression vectors, hostcells (including populations thereof), and antibodies (including antigenbinding portions thereof), can be isolated and/or purified. The term“isolated” as used herein means having been removed from its naturalenvironment. The term “purified” as used herein means having beenincreased in purity, wherein “purity” is a relative term, and not to benecessarily construed as absolute purity. For example, the purity can beat least about 50%, can be greater than 60%, 70%, 80%, 90%, 95%, or canbe 100%.

The inventive antigen recognizing constructs, TCRs, polypeptides,proteins (including functional variants thereof), nucleic acids,recombinant expression vectors, host cells (including populationsthereof), and antibodies (including antigen binding portions thereof),all of which are collectively referred to as “inventive TCR materials”hereinafter, can be formulated into a composition, such as apharmaceutical composition. In this regard, the invention provides apharmaceutical composition comprising any of the antigen recognizingconstructs, TCRs, polypeptides, proteins, functional portions,functional variants, nucleic acids, expression vectors, host cells(including populations thereof), and antibodies (including antigenbinding portions thereof) described herein, and a pharmaceuticallyacceptable carrier, excipient and/or stabilizer. The inventivepharmaceutical compositions containing any of the inventive TCRmaterials can comprise more than one inventive TCR material, e.g., apolypeptide and a nucleic acid, or two or more different TCRs (includingfunctional portions and functional variants thereof). Alternatively, thepharmaceutical composition can comprise an inventive TCR material incombination with another pharmaceutically active agent(s) or drug(s),such as chemotherapeutic agents, e.g., asparaginase, busulfan,carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil,gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab,vinblastine, vincristine, etc. Preferably, the carrier is apharmaceutically acceptable carrier. With respect to pharmaceuticalcompositions, the carrier can be any of those conventionally used forthe particular inventive TCR material under consideration. Suchpharmaceutically acceptable carriers are well-known to those skilled inthe art and are readily available to the public. It is preferred thatthe pharmaceutically acceptable carrier be one, which has no detrimentalside effects or toxicity under the conditions of use.

Thus also provided is a pharmaceutical composition, comprising any ofthe herein described products of the invention and TCR materials of theinvention, specifically any proteins, nucleic acids or host cells. In apreferred embodiment the pharmaceutical composition is for immunetherapy, preferably adoptive cell therapy.

Preferably, the inventive TCR material is administered by injection,e.g., intravenously. When the inventive TCR material is a host cellexpressing the inventive TCR (or functional variant thereof), thepharmaceutically acceptable carrier for the cells for injection mayinclude any isotonic carrier such as, for example, normal saline (about0.90% w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott,Chicago, Ill.), PLASMA-LYTE A (Baxter, Deerfield, Ill.), about 5%dextrose in water, or Ringer's lactate. In an embodiment, thepharmaceutically acceptable carrier is supplemented with human serumalbumen.

For purposes of the invention, the amount or dose (e.g., numbers ofcells when the inventive TCR material is one or more cells) of theinventive TCR material administered may be sufficient to affect, e.g., atherapeutic or prophylactic response, in the subject or animal over areasonable time frame. For example, the dose of the inventive TCRmaterial should be sufficient to bind to a cancer antigen, or detect,treat or prevent cancer in a period of from about 2 hours or longer,e.g., 12 to 24 or more hours, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular inventive TCR materialand the condition of the animal (e.g., human), as well as the bodyweight of the animal (e.g., human) to be treated.

It is contemplated that the inventive pharmaceutical compositions, TCRs(including functional variants thereof), polypeptides, proteins, nucleicacids, recombinant expression vectors, host cells, or populations ofcells can be used in methods of treating or preventing cancer, orCOL6A3-positive premalignancy. The inventive TCRs (and functionalvariants thereof) are believed to bind specifically to COL6A3 antigen,such that the TCR (or related inventive polypeptide or protein andfunctional variants thereof), when expressed by a cell, is able tomediate an immune response against a target cell expressing the COL6A3antigens of the invention. In this regard, the invention provides amethod of treating or preventing a condition, in particular cancer, in amammal, comprising administering to the mammal any of the pharmaceuticalcompositions, antigen recognizing constructs, in particular TCRs (andfunctional variants thereof), polypeptides, or proteins describedherein, any nucleic acid or recombinant expression vector comprising anucleotide sequence encoding any of the TCRs (and functional variantsthereof), polypeptides, proteins described herein, or any host cell orpopulation of cells comprising a recombinant vector, which encodes anyof the constructs of the invention (and functional variants thereof),polypeptides, or proteins described herein, in an amount effective totreat or prevent the condition in the mammal, wherein the condition iscancer, preferably COL6A3 positive cancer.

Examples of pharmaceutically acceptable carriers or diluents useful inthe present invention include stabilizers such as SPGA, carbohydrates(e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran), proteinssuch as albumin or casein, protein containing agents such as bovineserum or skimmed milk and buffers (e.g. phosphate buffer).

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof a condition in a mammal. Furthermore, the treatment or preventionprovided by the inventive method can include treatment or prevention ofone or more conditions or symptoms of the condition, e.g., cancer, beingtreated or prevented. For example, treatment or prevention can includepromoting the regression of a tumor. Also, for purposes herein,“prevention” can encompass delaying the onset of the condition, or asymptom or condition thereof.

The present invention also relates to a method of treating cancercomprising administering a TCR, a nucleic acid, or a host cell of thepresent description in combination with at least one chemotherapeuticagent and/or radiation therapy.

Also provided is a method of treating cancer in a subject in needthereof, comprising:

-   -   a) isolating a cell from said subject;    -   b) transforming the cell with at least one vector encoding an        antigen recognizing construct of the present invention to        produce a transformed cell;    -   c) expanding the transformed cell to produce a plurality of        transformed cells; and    -   d) administering the plurality of transformed cells to said        subject.

Also provided is a method of treating cancer in a subject in needthereof, comprising:

-   -   a) isolating a cell from a healthy donor;    -   b) transforming the cell with a vector encoding an antigen        recognizing construct of the present invention to produce a        transformed cell;    -   c) expanding the transformed cell to produce a plurality of        transformed cells; and    -   d) administering the plurality of transformed cells to said        subject.

Also provided is a method of detecting cancer in a biological samplecomprising:

-   -   a) contacting the biological sample with an antigen recognizing        construct of the present description;    -   b) detecting binding of the antigen recognizing construct to the        biological sample.

In some embodiments, the method of detecting cancer is carried out invitro, in vivo or in situ.

Also provided is a method of detecting the presence of a condition in amammal. The method comprises (i) contacting a sample comprising one ormore cells from the mammal with any of the inventive TCRs (andfunctional variants thereof), polypeptides, proteins, nucleic acids,recombinant expression vectors, host cells, populations of cells,antibodies, or antigen binding portions thereof, or pharmaceuticalcompositions described herein, thereby forming a complex, and detectingthe complex, wherein detection of the complex is indicative of thepresence of the condition in the mammal, wherein the condition iscancer, such as a COL6A3-positive malignancy.

With respect to the inventive method of detecting a condition in amammal, the sample of cells can be a sample comprising whole cells,lysates thereof, or a fraction of the whole cell lysates, e.g., anuclear or cytoplasmic fraction, a whole protein fraction, or a nucleicacid fraction.

For purposes of the inventive detecting method, the contacting can takeplace in vitro or in vivo with respect to the mammal. Preferably, thecontacting is in vitro.

Also, detection of the complex can occur through any number of waysknown in the art. For instance, the inventive antigen recognizingconstructs (and functional variants thereof), polypeptides, proteins,nucleic acids, recombinant expression vectors, host cells, populationsof cells, or antibodies or TCRs, or antigen binding portions thereof,described herein, can be labeled with a detectable label such as, forinstance, a radioisotope, a fluorophore (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkalinephosphatase, horseradish peroxidase), and element particles (e.g., goldparticles).

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the mammal. Preferably, the cells are autologous to themammal.

With respect to the above mentioned medical applications of the TCRmaterial of the invention, the to be treated and/or diagnosed cancer canbe any cancer, including any of acute lymphocytic cancer, acute myeloidleukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breastcancer, cancer of the anus, anal canal, or anorectum, cancer of the eye,cancer of the intrahepatic bile duct, cancer of the joints, cancer ofthe neck, gallbladder, or pleura, cancer of the nose, nasal cavity, ormiddle ear, cancer of the oral cavity, cancer of the vagina, cancer ofthe vulva, chronic lymphocytic leukemia, chronic myeloid cancer, coloncancer, esophageal cancer, cervical cancer, gastrointestinal carcinoidtumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer,larynx cancer, liver cancer, lung cancer, malignant mesothelioma,melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma,cancer of the oropharynx, ovarian cancer, cancer of the penis,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, stomach cancer, testicular cancer,thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladdercancer. A preferred cancer is cancer is cancer of the uterine cervix,oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis. Aparticularly preferred cancer is a COL6A3 positive cancer, such asgastrointestinal or gastric cancer.

In general, the invention provides a method for treating a subjectsuffering from a tumor or tumor disease comprising the administration ofthe antigen recognizing constructs, nucleic acids, vectors,pharmaceutical compositions and/or host cell as disclosed by the presentinvention. Preferably the subject is a subject in need of such atreatment. The subject in preferred embodiments is a mammalian subject,preferably a human patient, suffering from a tumor or tumor disease,which is COL6A3-positive.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The present invention will now be further described in the followingexamples with reference to the accompanying figures and sequences,nevertheless, without being limited thereto. For the purposes of thepresent invention, all references as cited herein are incorporated byreference in their entireties. In the Figures and Sequences:

FIG. 1: IFNγ release (left axis) and HLA-A*02/COL6A3-002 tetramerstaining (right axis) of human primary CD8+ T-cells of one donorelectroporated with alpha and beta chain RNA of TCR R4P1D10 (Table 1),respectively, after co-incubation with K562-A2 target cells (see HiranoN. et al; Blood. 2006 Feb. 15; 107(4):1528-36) loaded with COL6A3-002peptide (SEQ ID NO:58), various COL6A3-002 alanine or glycinesubstitution variants at positions 1-9 of (SEQ ID NO:59-67), orNYESO1-001 control peptide (SEQ ID NO:68).

FIG. 2: IFNγ release (left axis) and HLA-A*02/COL6A3-002 tetramerstaining (right axis) of human primary CD8+ T-cells of one donorelectroporated with alpha and beta chain RNA of TCR R4P1D10 (Table 1),respectively, after co-incubation with K562-A2 target cells loaded withCOL6A3-002 peptide (SEQ ID NO:58), homologous but unrelated peptideAGRN-001, CLASP-001, COL6A1-001, COL6A2-001, COL6A3-006, COL6A3-008,COL6A3-014, VWA2-001, VWF-001 (SEQ ID NO:49-57) or NYESO1-001 controlpeptide (SEQ ID NO:68). Electroporated CD8+ T-cells only (E only) serveas control.

FIG. 3: HLA-A*02/COL6A3-002 tetramer and HLA-A*02/NYESO1-001 tetramerstaining, respectively, of J.RT3-T3.5 cells electroporated with alphaand beta chain RNA of TCR R4P1D10 or NYESO1-001-specific control TCR 1G4(Table 1). Mock electroporated J.RT3-T3.5 cells serve as control.

FIG. 4: HLA-A*02/COL6A3-002 tetramer and HLA-A*02/NYESO1-001 tetramerstaining, respectively, of SUP-T1 cells electroporated with alpha andbeta chain RNA of TCR R4P1D10 or NYESO1-001-specific control TCR 1G4(Table 1). Mock electroporated SUP-T1 cells serve as control.

FIG. 5: HLA-A*02/COL6A3-002 tetramer and HLA-A*02/NYESO1-001 tetramerstaining, respectively, of human primary CD8+ T-cells of one donorelectroporated with alpha and beta chain RNA of TCR R4P1D10 orNYESO1-001-specific control TCR 1G4 (Table 1). Mock electroporated CD8+T-cells serve as control.

FIG. 6: IFNγ release of human primary CD8+ T-cells of one donorelectroporated with alpha and beta chain RNA of TCR R4P3F9 (Table 1),respectively, after co-incubation with K562-A2 target cells loaded withCOL6A3-002 peptide (SEQ ID NO:58), various COL6A3-002 alanine or glycinesubstitution variants at positions 1-9 of (SEQ ID NO:59-67), orNYESO1-001 control peptide (SEQ ID NO:68).

FIG. 7: IFNγ release of human primary CD8+ T-cells of one donorelectroporated with alpha and beta chain RNA of TCR R4P3F9 (Table 1),respectively, after co-incubation with K562-A2 target cells loaded withCOL6A3-002 peptide (SEQ ID NO:58), homologous but unrelated peptideAGRN-001, CLASP-001, COL6A1-001, COL6A2-001, COL6A3-006, COL6A3-008,COL6A3-014, VWA2-001, VWF-001 (SEQ ID NO:49-57) or NYESO1-001 controlpeptide (SEQ ID NO:68). Mock electroporated CD8+ T-cells (E only) serveas control.

FIG. 8: HLA-A*02/COL6A3-002 tetramer and HLA-A*02/NYESO1-001 tetramerstaining, respectively, of J.RT3-T3.5 cells electroporated with alphaand beta chain RNA of TCR R4P3F9 or NYESO1-001-specific control TCR 1G4(Table 1). Mock electroporated J.RT3-T3.5 cells serve as control.

FIG. 9: HLA-A*02/COL6A3-002 tetramer and HLA-A*02/NYESO1-001 tetramerstaining, respectively, of SUP-T1 cells electroporated with alpha andbeta chain RNA of TCR R4P3F9 or NYESO1-001-specific control TCR 1G4(Table 1). Mock electroporated SUP-T1 cells serve as control.

FIG. 10: IFNγ release of human primary CD8+ T-cells of one donorelectroporated with alpha and beta chain RNA of TCR R4P3H3 (Table 1),respectively, after co-incubation with K562-A2 target cells loaded withCOL6A3-002 peptide (SEQ ID NO:58), various COL6A3-002 alanine or glycinesubstitution variants at positions 1-9 of (SEQ ID NO:59-67) orNYESO1-001 control peptide (SEQ ID NO:68).

FIG. 11: IFNγ release of human primary CD8+ T-cells of one donorelectroporated with alpha and beta chain RNA of TCR R4P3H3 (Table 1),respectively, after co-incubation with K562-A2 target cells loaded withCOL6A3-002 peptide (SEQ ID NO:58), homologous but unrelated peptideAGRN-001, CLASP-001, COL6A1-001, COL6A2-001, COL6A3-006, COL6A3-008,COL6A3-014, VWA2-001, VWF-001 (SEQ ID NO:49-57) or NYESO1-001 controlpeptide (SEQ ID NO:68). Mock electroporated CD8+ T-cells (E only) serveas control.

FIG. 12: HLA-A*02/COL6A3-002 tetramer and HLA-A*02/NYESO1-001 tetramerstaining, respectively, of SUP-T1 cells electroporated with alpha andbeta chain RNA of TCR R4P3H3 or NYESO1-001-specific control TCR 1G4(Table 1). Mock electroporated SUP-T1 cells serve as control.

DETAILED DESCRIPTION OF THE INVENTION

TABLE 1 TCR sequences of the invention SEQ ID NO: TCR Chain RegionSequence  1 R4P1D10 alpha CDR1 DRGSQS  2 R4P1D10 alpha CDR2 IY  3R4P1D10 alpha CDR3 CAVNFHDKIIF  4 R4P1D10 alpha variable domainMKSLRVLLVILWLQLSWVWSQQKE VEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGD KEDGRFTAQLNKASQYVSLLIRDSQ PSDSATYLCAVN  5R4P1D10 alpha constant domain NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLD MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEK SFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS  6R4P1D10 alpha full-length MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRG SQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ PSDSATYLCAVNFHDKIIFGKGTRLHILPNIQNPDPAVYQLRDSKSSDKSVC LFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFA CANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLL KVAGFNLLMTLRLWSS  7 R4P1D10 beta CDR1 SGDLS 8 R4P1D10 beta CDR2 YYNGEE  9 R4P1D10 beta CDR3 CASSVASAYGYTF 10R4P1D10 beta variable domain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSV YWYQQSLDQGLQFLIHYYNGEERAKGNILERFSAQQFPDLHSELNLSSLE LGDSALYFCASSV 11 R4P1D10 beta constant domainEDLNKVFPPEVAVFEPSEAEISHTQK ATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKR KDF 12 R4P1D10 beta full-lengthMGFRLLCCVAFCLLGAGPVDSGVTQ TPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIHYYNGEERA KGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVASAYGYTFGSGT RLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSW WVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHF RCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVL SATILYEILLGKATLYAVLVSALVLM AMVKRKDF 13 R4P3F9alpha CDR1 DRGSQS 14 R4P3F9 alpha CDR2 IY 15 R4P3F9 alpha CDR3CAAYSGAGSYQLTF 16 R4P3F9 alpha variable domain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRG SQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ PSDSATYLCA 17 R4P3F9 alpha constant domainNIQNPDPAVYQLRDSKSSDKSVCLFT DFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 18 R4P3 F9 alpha full-lengthMKSLRVLLVILWLQLSWVWSQQKE VEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGD KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLTFGKG TKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT DKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCD VKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 19 R4P3F9 beta CDR1 SGDLS 20 R4P3F9 beta CDR2 YYNGEE21 R4P3F9 beta CDR3 CASSVESSYGYTF 22 R4P3F9 beta variable domainMGFRLLCCVAFCLLGAGPVDGVTQ TPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIHYYNGEERA KGNILERFSAQQFPDLHSELNLSSLE LGDSALYFCASSV 23R4P3F9 beta constant domain EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGK EVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQF YGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYE ILLGKATLYAVLVSALVLMAMVKR KDF 24 R4P3F9 betafull-length MGFRLLCCVAFCLLGAGPVDSGVTQ TPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERA KGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSGTR LTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWW VNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRC QVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSA TILYEILLGKATLYAVLVSALVLMA MVKRKDF 25 R4P3H3alpha CDR1 DRGSQS 26 R4P3H3 alpha CDR2 IY 27 R4P3H3 alpha CDR3CAVKAGNQFYF 28 R4P3H3 alpha variable domain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRG SQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ PSDSATYLCAV 29 R4P3H3 alpha constant domainNIQNPDPAVYQLRDSKSSDKSVCLFT DFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 30 R4P3H3 alpha full-lengthMKSLRVLLVILWLQLSWVWSQQKE VEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGD KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKAGNQFYFGTGTSL TVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKT VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKL VEKSFETDTNLNFQNLSVIGFRILLL KVAGFNLLMTLRLWSS31 R4P3H3 beta CDR1 SGHVS 32 R4P3H3 beta CDR2 FQNEAQ 33 R4P3H3 beta CDR3CASSLLTSGGDNEQFF 34 R4P3H3 beta variable domain MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHV SLFWYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQR TQQEDSAVYLCASSL 35 R4P3H3 betaconstant domain EDLKNVFPPEVAVFEPSEAEISHTQK ATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG 36 R4P3H3 beta full-lengthMGTRLLCWVVLGFLGTDHTGAGVS QSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEAQL DKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSLLTSGGDNEQF FGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVE LSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPR NHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQ GVLSATILYEILLGKATLYAVLVSAL VLMAMVKRKDSRG 371G4 alpha CDR1 DSAIYN 38 1G4 alpha CDR2 IQS 39 1G4 alpha CDR3CAVRPTSGGSYIPTF 40 1G4 alpha variable domain METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYN LQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGD SATYLCAVR 41 1G4 alpha constant domainYIQNPDPAVYQLRDSKSSDKSVCLFT DFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 42 1G4 alpha full-lengthMETLLGLLILWLQLQWVSSKQEVTQ IPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQT SGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLI VHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKT VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKL VEKSFETDTNLNFQNLSVIGFRILLL KVAGFNLLMTLRLWSS43 1G4 beta CDR1 MNHEY 44 1G4 beta CDR2 SVGAGI 45 1G4 beta CDR3CASSYVGNTGELFF 46 1G4 beta variable domain MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHE YMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLS AAPSQTSVYFCASSY 47 1G4 beta constant domainEDLKNVFPPEVAVFEPSEAEISHTQK ATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG 48 1G4 beta fulllengthMSIGLLCCAALSLLWAGPVNAGVTQ TPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGI TDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFG EGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELS WWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNH FRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGV LSATILYEILLGKATLYAVLVSALVL MAMVKRKDSRG

TABLE 2 Peptide sequences of the invention Peptide Code SequenceSEQ ID NO: AGRN-001 ALLDGRVQL 49 CLASP1-001 RLLDGAFKL 50 COL6A1-001ILLDGSASV 51 COL6A2-001 FLLDGSERL 52 COL6A3-006 FLFDGSANLV 53 COL6A3-008FLFDGSANL 54 COL6A3-014 FLLDGSEGV 55 VWA2-001 FLLDGSNSV 56 VWF-001FLLDGSSRL 57 COL6A3-002 FLLDGSANV 58 A1 ALLDGSANV 59 A2 FALDGSANV 60 A3FLADGSANV 61 A4 FLLAGSANV 62 AS FLLDASANV 63 A6 FLLDGAANV 64 A7FLLDGSGNV 65 A8 FLLDGSAAV 66 A9 FLLDGSANA 67 NYESO1-001 SLLMWITQV 68

EXAMPLES

In an aspect, allo-reactive settings are used to circumventself-tolerance and yield T-cells with a higher avidity when compared toT-cells derived from autologous settings, i.e., patients. Examples ofsuch settings include in vitro generation of allo-HLA reactive,peptide-specific T-cells (Sadovnikova et al. 1998; Savage et al. 2004;Wilde et al. 2012), and immunization of mice transgenic for human-MHC orhuman TCR (Stanislawski et al. 2001; Li et al. 2010), each of which areincorporated by reference in their entireties.

To isolate high avidity T-cells from allo-reactive setting, PBMCs fromHLA-A*02-negative healthy donors are used after obtaining informedconsent. Recombinant biotinylated HLA-A*02 class I monomers and A2fluorescent tetramers containing COL6A3-002 are obtained from MBLI(Woburn, Mass.). PBMCs are incubated with anti-CD20SA diluted inphosphate buffered saline (PBS) for 1 hour at room temperature, washed,and incubated with the biotinylated HLA-A*02/COL6A3-002 monomers for 30minutes at room temperature, washed, and plated at 3×10⁶ cells/well in24-well plates in RPMI with 10% human AB serum. Interleukin 7 (IL-7; R&DSystems, Minneapolis, Minn.) was added on day 1 at 10 ng/mL and IL-2(Chiron, Harefield, United Kingdom) was added at 10 U/mL on day 4. Overa 5-week period cells were restimulated weekly with fresh PBMCs, mixedwith responder cells at a 1:1 ratio, and plated at 3×10⁶/well in 24-wellplates.

To obtain high avidity T-cells, approximately 10⁶ PBMCs withHLA-A*02/COL6A3-002 tetramer-phycoerythrin (PE) (obtained from MBLI)were incubated for 30 minutes at 37° C., followed byanti-CD8-fluorescein isothiocyanate (FITC)/allophycocyanin (APC) for 20minutes at 4° C., followed by fluorescence activated cell sorting.Sorted tetramer-positive cells were expanded in 24-well plates using,per well, 2×10⁵ sorted cells, 2×10⁶ irradiated A2-negative PBMCs asfeeders, 2×10⁴ CD3/CD28 beads/mL (Dynal, Oslo Norway), and IL-2 (1000U/mL). The high avidity T-cells, thus obtained, were then used toidentify and isolate TCRs using techniques known in the art, such assingle cell 5′ RACE (Rapid Amplification of cDNA Ends). Non-redundantTCR DNAs were then analyzed for amino acid/DNA sequences determinationand cloning into expression vectors.

Three COL6A3-002-specific TCRs (R4P1D10, R4P3F9 and R4P3H3, see Table2), each encoding tumor specific TCR-alpha and TCR-beta chains, wereisolated and amplified from T-cells of healthy donors. Cells fromhealthy donors were in vitro stimulated according to a method previouslydescribed (Walter et al., 2003 J Immunol., November 15; 171(10):4974-8).COL6A3 peptide presentation was performed as described previously(Hirano N. et al; Blood. 2006 Feb. 15; 107(4):1528-36). Target-specificcells were single-cell sorted using HLA-A*02 multimers and then used forsubsequent TCR isolation. TCR sequences were isolated via 5′ RACE bystandard methods as described by e.g. Molecular Cloning a laboratorymanual fourth edition by Green and Sambrook. The alpha and beta variableregions of TCRs R4P1D10, R4P3F9 and R4P3H3 were sequenced and cloned forfurther functional characterization. R4P1D10 and R4P3H3 are derived fromHLA-A*02 positive donors and R4P3F9 is derived from a HLA-A*02 negativedonor (allo-reactive setting).

TABLE 3 SPR affinity of COL6A3-002 and NYESO1-001 TCRs Equilibriumdissociation Equilibrium dissociation constant (K_(D)) for HLA-A02/constant (K_(D)) for HLA-A02/ TCR COL6A3-002 complex in μM NYESO1-001complex in μM R4P1D10 16 no binding R4P3F9 62 no binding R4P3H3 102 nobinding 1G4 no binding 7

Example 1: T-Cell Receptor R4P1D10

The TCR R4P1D10 alpha and beta chains were cloned as describedpreviously, for example, as described in U.S. Pat. No. 8,519,100, thecontent of which is hereby incorporated by reference in its entirety forsaid methods. TCR R4P1D10 is restricted towards HLA-A2-presentedCOL6A3-002 (see table 3 above).

TABLE 4 Features of R4P1D10 alpha chain: Start Stop Description Sequence  1  21 L segment MKSLRVLLVILWLQLSWVWSQ (SEQ ID NO: 69) (TRAV12-2)   1113 V chain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPL (TRAV12-2)SVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSP ELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVN  48  53 CDR1 DRGSQS  71  72 CDR2 IY 110 120 CDR3CAVNFHDKIIF 116 130 J segment DKIIFGKGTRLHILP (TRAJ30) 131 272 ConstantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTN regionVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAW (TRAC)SNKSDFACANAFNNSIIPEDTFFPSPESSCDVKL VEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

TABLE 5 Features of R4P1D10 beta chain: Start Stop Description Sequence  1  19 L segment MGFRLLCCVAFCLLGAGPV (SEQ ID NO: 70) (TRBV9)   1 114V chain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLIT (TRBV9)ATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQ FLIHYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSV  46  50 CDR1 SGDLS  68  73 CDR2 YYNGEE 110 122 CDR3CASSVASAYGYTF 118 131 J chain YGYTFGSGTRLTVV (TRBJ1-2) 132 308 constantEDLNKVFPPEVAVFEPSEAEISHTQKATLVCL region ATGFFPDHVELSWWVNGKEVHSGVSTDPQPL(TRBC1) KEQPALNDSRYCLSSRLRVSATFWQNPRNHF RCQVQFYGLSENDEWTQDRAKPVTQIV AEAWGRADCGFTSVSYQQGVLSATILYEILLGKAT LYAVLVSALVLMAMVKRKDF

R4P1D10 specifically recognizes COL6A3-002 as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells and bind HLA-A*02 tetramers, respectively, loaded eitherwith COL6A3-002 peptide or alanine and glycine substitution variants ofCOL6A3-002 (FIG. 1) or different peptides showing high degree ofsequence similarity to COL6A3-002 (FIG. 2). NYESO1-001 peptide is usedas negative control.

Re-expression of R4P1D10 leads to selective binding ofHLA-A*02/COL6A3-002 tetramers but not HLA-A*02/NYESO1-001 tetramers inJ.RT3-T3.5 Jurkat cells (FIG. 3), SUP-T1 cells (FIG. 4) and humanprimary CD8+ T-cells (FIG. 5). For each cell type, re-expression of theNYESO1-001-specific TCR 1G4 and mock expression are used as control.

SPR (Surface Plasmon Resonance) binding analysis for R4P1D10, expressedas soluble TCR according to a previously described method (Willcox B Eet al., 1999 Protein Sci., November; 8(11):2418-23), andHLA-A*02/COL6A3-002 complex reveals an affinity of K_(D)=16 μM (Table3). SPR binding data for 1G4 TCR and HLA-A*02/NYESO1-001 are used ascontrol.

Example 2: T-Cell Receptor R4P3F9

The TCR R4P3F9 alpha and beta chains were cloned as described before,for example, as described in U.S. Pat. No. 8,519,100, which is herebyincorporated by reference in its entirety for said methods. TCR R4P3F9is restricted towards HLA-A2-presented COL6A3-002 (see table 3 above).

TABLE 6 Features of R4P3F9 alpha chain Start Stop Description Sequence  1  21 L segment MKSLRVLLVILWLQLSWVWSQ (SEQ ID NO: 71) (TRAV12-2)   1111 V chain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPL (TRAV12-2)SVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSP ELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCA  48  53 CDR1 DRGSQS  71  72 CDR2 IY 110 123 CDR3CAAYSGAGSYQLTF 113 133 J segment YSGAGSYQLTFGKGTKLSVIP (TRAJ28) 134 274Constant NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTN regionVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAW (TRAC)SNKSDFACANAFNNSIIPEDTFFPSPESSCDVKL VEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

TABLE 7 Features of R4P3F9 beta chain Start Stop Description Sequence  1  19 L segment MGFRLLCCVAFCLLGAGPV (SEQ ID NO: 72) (TRBV9)   1 114V chain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLIT (TRBV9)ATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQ FLIHYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSV  46  50 CDR1 SGDLS  68  73 CDR2 YYNGEE 110 122 CDR3CASSVESSYGYTF 118 131 J chain YGYTFGSGTRLTVV (TRBJ1-2) 132 308 constantEDLNKVFPPEVAVFEPSEAEISHTQKATLVCL region ATGFFPDHVELSWWVNGKEVHSGVSTDPQPL(TRBC1) KEQPALNDSRYCLSSRLRVSATFWQNPRNHF RCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKAT LYAVLVSALVLMAMVKRKDFR4P3F9 specifically recognizes COL6A3-002 as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells, respectively, loaded either with COL6A3-002 peptide oralanine and glycine substitution variants of COL6A3-002 (FIG. 6) ordifferent peptides showing high degree of sequence similarity toCOL6A3-002 (FIG. 7). NYESO1-001 peptide is used as negative control.

Re-expression of R4P3F9 leads to selective binding ofHLA-A*02/COL6A3-002 tetramers but not HLA-A*02/NYESO1-001 tetramers inJ.RT3-T3.5 Jurkat cells (FIG. 8) and SUP-T1 cells (FIG. 9). For eachcell type, re-expression of the NYESO1-001-specific TCR 1G4 and mockexpression are used as control.

SPR binding analysis for R4P3F9, expressed as soluble TCR according to apreviously described method (Willcox B E et al., 1999 Protein Sci.,November; 8(11):2418-23), and HLA-A*02/COL6A3-002 complex reveals anaffinity of K_(D)=62 μM (Table 3). SPR binding data for 1G4 TCR andHLA-A*02/NYESO1-001 are used as control.

Example 3: T-Cell Receptor R4P3H3

The TCR R4P3H3 alpha and beta chains were cloned as described before,for example, as described in U.S. Pat. No. 8,519,100, which is herebyincorporated by reference in its entirety. TCR R4P3H3 is restrictedtowards HLA-A2-presented COL6A3-002 (see table 3 above).

TABLE 8 Features of R4P3H3 alpha chain Start Stop Description Sequence  1  21 L segment MKSLRVLLVILWLQLSWVWSQ (SEQ ID NO: 73) (TRAV12-2)   1112 V chain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPL (TRAV12-2)SVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSP ELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAV  48  53 CDR1 DRGSQS  71  72 CDR2 IY 110 120 CDR3CAVKAGNQFYF 115 130 J segment GNQFYFGTGTSLTVIP (TRAJ49) 131 271 ConstantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTN regionVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAW (TRAC)SNKSDFACANAFNNSIIPEDTFFPSPESSCDVKL VEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

TABLE 9 Features of R4P3H3 beta chain Start Stop Description Sequence  1  19 L segment MGTRLLCWVVLGFLGTDHT (SEQ ID NO: 74) (TRB V7-8)   1 115V chain MGTRLLCWVVLGFLGTDHTGAGVSQSPRYK (TRB V7-8)VAKRGQDVALRCDPISGHVSLFWYQQALGQ GPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSL  46  50 CDR1 SGHVS  68  73 CDR2 FQNEAQ 111 126CDR3 CASSLLTSGGDNEQFF 122 135 J chain NEQFFGPGTRLTVL (TRBJ2-1) 136 314constant EDLKNVFPPEVAVFEPSEAEISHTQKATLVCL regionATGFYPDHVELSWWVNGKEVHSGVSTDPQP (TRBC2) LKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAE AWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

R4P3H3 specifically recognizes COL6A3-002 as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells, respectively, loaded either with COL6A3-002 peptide oralanine and glycine substitution variants of COL6A3-002 (FIG. 10) ordifferent peptides showing high degree of sequence similarity toCOL6A3-002 (FIG. 11). NYESO1-001 peptide is used as negative control.

Re-expression of R4P3H3 leads to selective binding ofHLA-A*02/COL6A3-002 tetramers but not HLA-A*02/NYESO1-001 tetramers inSUP-T1 cells (FIG. 12). Re-expression of the NYESO1-001-specific TCR 1G4and mock expression are used as control.

SPR binding analysis for R4P3H3, expressed as soluble TCR according to apreviously described method (Willcox B E et al., 1999 Protein Sci.,November; 8(11):2418-23), and HLA-A*02/COL6A3-002 complex reveals anaffinity of K_(D)=102 μM (Table 3). SPR binding data for 1G4 TCR andHLA-A*02/NYESO1-001 are used as control.

The invention claimed is:
 1. A single chain TCR comprising the aminoacid sequences of (a) SEQ ID NOs: 1, 2, 3, 7, 8, and 9; or (b) SEQ IDNOs: 13, 14, 15, 19, 20, and 21, or (c) SEQ ID NOs: 25, 26, 27, 31, 32,and
 33. 2. The single chain TCR of claim 1, wherein the TCR variablechain region consists of the amino acid sequence of SEQ ID NO:
 4. 3. Thesingle chain TCR of claim 1, comprising a CDR1α chain comprising theamino acid sequence of SEQ ID NO: 1, a CDR2α chain comprising the aminoacid sequence of SEQ ID NO: 2, a CDR3α chain comprising the amino acidsequence of SEQ ID NO: 3, a CDR1β chain comprising the amino acidsequences of SEQ ID NO: 7, a CDR2β chain comprising the amino acidsequence of SEQ ID NO: 8, and a CDR3β chain comprising the amino acidsequence of SEQ ID NO: 9; or a CDR1α chain comprising the amino acidsequence of SEQ ID NO: 13, a CDR2α chain comprising the amino acidsequence of SEQ ID NO: 14, a CDR3α chain comprising the amino acidsequence of SEQ ID NO: 15, a CDR1β chain comprising the amino acidsequences of SEQ ID NO: 19, a CDR2β chain comprising the amino acidsequence of SEQ ID NO: 20, and a CDR3β chain comprising the amino acidsequence of SEQ ID NO: 21; or a CDR1α chain comprising the amino acidsequence of SEQ ID NO: 25, a CDR2α chain comprising the amino acidsequence of SEQ ID NO: 26, a CDR3α chain comprising the amino acidsequence of SEQ ID NO: 27, a CDR1β chain comprising the amino acidsequence of SEQ ID NO: 31, a CDR2β chain comprising the amino acidsequence of SEQ ID NO: 32, and a CDR3β chain comprising the amino acidsequence of SEQ ID NO:
 33. 4. The single chain TCR of claim 1,comprising a CDR1α chain consisting of the amino acid sequence of SEQ IDNO: 1, a CDR2α chain comprising the amino acid sequence of SEQ ID NO: 2,a CDR3α chain comprising the amino acid sequence of SEQ ID NO: 3, aCDR1β chain consisting of the amino acid sequences of SEQ ID NO: 7, aCDR2β chain comprising the amino acid sequence of SEQ ID NO: 8, and aCDR3β chain comprising the amino acid sequence of SEQ ID NO: 9; or aCDR1α chain consisting of the amino acid sequence of SEQ ID NO: 13, aCDR2α chain comprising the amino acid sequence of SEQ ID NO: 14, a CDR3αchain comprising the amino acid sequence of SEQ ID NO: 15, a CDR1β chainconsisting of the amino acid sequences of SEQ ID NO: 19, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 20, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO: 21; or a CDR1α chainconsisting of the amino acid sequence of SEQ ID NO: 25, a CDR2α chaincomprising the amino acid sequence of SEQ ID NO: 26, a CDR3α chaincomprising the amino acid sequence of SEQ ID NO: 27, a CDR1β chainconsisting of the amino acid sequence of SEQ ID NO: 31, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 32, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO:
 33. 5. The single chainTCR of claim 1, comprising a CDR1α chain comprising the amino acidsequence of SEQ ID NO: 1, a CDR2α chain consisting of the amino acidsequence of SEQ ID NO: 2, a CDR3α chain comprising the amino acidsequence of SEQ ID NO: 3, a CDR1β chain comprising the amino acidsequences of SEQ ID NO: 7, a CDR2β chain consisting of the amino acidsequence of SEQ ID NO: 8, and a CDR3β chain comprising the amino acidsequence of SEQ ID NO: 9; or a CDR1α chain comprising the amino acidsequence of SEQ ID NO: 13, a CDR2α chain consisting of the amino acidsequence of SEQ ID NO: 14, a CDR3α chain comprising the amino acidsequence of SEQ ID NO: 15, a CDR1β chain comprising the amino acidsequences of SEQ ID NO: 19, a CDR2β chain consisting of the amino acidsequence of SEQ ID NO: 20, and a CDR3β chain comprising the amino acidsequence of SEQ ID NO: 21; or a CDR1α chain comprising the amino acidsequence of SEQ ID NO: 25, a CDR2α chain consisting of the amino acidsequence of SEQ ID NO: 26, a CDR3α chain comprising the amino acidsequence of SEQ ID NO: 27, a CDR1β chain comprising the amino acidsequence of SEQ ID NO: 31, a CDR2β chain consisting of the amino acidsequence of SEQ ID NO: 32, and a CDR3β chain comprising the amino acidsequence of SEQ ID NO:
 33. 6. The single chain TCR of claim 1,comprising a CDR1α chain consisting of the amino acid sequence of SEQ IDNO: 1, a CDR2α chain comprising the amino acid sequence of SEQ ID NO: 2,a CDR3α chain comprising the amino acid sequence of SEQ ID NO: 3, aCDR1β chain consisting of the amino acid sequences of SEQ ID NO: 7, aCDR2β chain comprising the amino acid sequence of SEQ ID NO: 8, and aCDR3β chain comprising the amino acid sequence of SEQ ID NO:
 9. 7. Thesingle chain TCR of claim 1, comprising a CDR1α chain comprising theamino acid sequence of SEQ ID NO: 1, a CDR2α chain consisting of theamino acid sequence of SEQ ID NO: 2, a CDR3α chain comprising the aminoacid sequence of SEQ ID NO: 3, a CDR1β chain comprising the amino acidsequences of SEQ ID NO: 7, a CDR2β chain consisting of the amino acidsequence of SEQ ID NO: 8, and a CDR3β chain comprising the amino acidsequence of SEQ ID NO:
 9. 8. The single chain TCR of claim 1, comprisinga CDR1α chain comprising the amino acid sequence of SEQ ID NO: 1, aCDR2α chain comprising the amino acid sequence of SEQ ID NO: 2, a CDR3αchain consisting of the amino acid sequence of SEQ ID NO: 3, a CDR1βchain comprising the amino acid sequences of SEQ ID NO: 7, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 8, and a CDR3β chainconsisting of the amino acid sequence of SEQ ID NO: 9.